This proposal addresses HPV mucosal vaccination in the context of different mucosal adjuvants to further elucidate the type (secretory, humoral, cellular), extent (titer, number of antibody secreting B cells) and quality (antibody avidity, virus neutralization titer) of the induced response. As indicated in preliminary studies, HPV-11 VLP, administered vaginally plus intramuscularly, can induce a secretory immune response with the potential to mediate in vitro neutralization of HPV-11 infectivity. Both animals mounted a measurable response within vaginal secretions but only one animal was able to mediate this type of neutralization. Previous studies (1) have demonstrated that monoclonal antibody-mediated neutralization of HPV-11 in vitro infectivity corresponds to prevention of condyloma formation in the nude mouse xenograft system (2). This data suggests that VLP in the presence of an appropriate mucosal adjuvant will induce a high titer neutralizing antibody response within mucosal tissues. Although several studies have used the nontoxic B subunit of cholera toxin (CTB), a mucosal adjuvant, for enhanced mucosal immunity in rhesus macaques more recent data suggests it may be less than optimal. One study reported induction of significant secretory immune responses following immunization of rhesus macaques with SIV p27:Ty-VLP-CTB conjugates by oral-vaginal-rectal immunization routes. However, recent challenge experiments with SIV in these animals indicate the response was not of significant quality to protect from challenge with infectious SIV. Conversely, ongoing studies at the California Regional Primate Research Center indicate cholera toxin (CT) may serve as a more effective mucosal adjuvant within the rhesus model due to the nature of CT being a strong mucosal adjuvant in these animals without the concomitant toxic effects observed in humans (C.J. Miller, Appendix C). This study will help to dissect out which components of mucosal and systemic immunity to HPV-VLP are important for induction of a strong protective response within the genital and aero-digestive tracts in a non-human primate model. This should further promote the rational design of vaccine trials in humans (and requirement for mucosal adjuvant) to induce both protective and multi-type immunity within mucosal tissues. The goal of this study is to establish a gold standard of mucosal immune response to VLP antigens in a non-human primate against which other HPV vaccine formulations can be compared in order to optimize HPV vaccinology.